Method of producing L-lysine by fermentation

ABSTRACT

L-lysine is accumulated by -L-alanine, an L-lysine-producing mutant which belongs to one of the genera Brevibacterium, Corynebacterium and Arthrobacter and is resistant to one of the metabolic antagonists consisting of A. L-lysine hydroxamate, B. S-( Beta -aminoethyl)-L-cysteine hydroxamate, C. 3-( Beta -AMINOETHYL-SULFINYL)-L-alanine (S-dl), D. 3-(S-methyl-S-( Beta -aminoethyl)-thio)-L-alamine, E. S- Beta -(2-pyridyl)ethyl-L-cysteine, and F. S- Beta -(4-pyridyl)ethyl-L-cysteine, IN A MEDIUM.

United States Patent Kurimura et al.

[4 1 Sept. 16, 1975 [73] Assignee: Mitsui Toatsu Chemicals,

Incorporated, Tokyo, Japan [22] Filed: Jan. 17, 1973 [21] Appl. No.: 324,319

[30] Foreign Application Priority Data Jan. 21, I972 Japan 47-7632 Jan. 21, 1972 Japan.... 477633 Jan. 2l, l972 Japan 47-7634 [52] U.S. Cl 195/28 R; 195/29; 195/47; 195/49 [51] Int. Cl. C12D 13/06 [58] Field of Search 195/28 R, 29, 47, 112, '195/49 [56] References Cited UNITED STATES PATENTS 3,595,751 7/1971 Nakayama et al. l95/29 X 3,6l6,2l8 lO/l97l Shiio et al 195/29 3,708,395 l/l973 Nakayama et a]... 195/112 X 3,711,374 l/1973 Tanaka et al. l95/28 R OTHER PUBLlCATlONS Bekers et al., Production of Lysine Feed Concentrates, cited in Chemical Abstracts 73:97355n.

Primary ExaminrLionel M. Shapiro Assistant ExaminerR. B. Penland Attorney, Agent, or FirmBrowdy and Neimark [5 7] ABSTRACT L-lysine is accumulated by -L-alanine, an L-lysineproducing mutant which belongs to one of the genera Brevibacterium, Corynebacterium and Arthrobacter and is resistant to one of the metabolic antagonists consisting of a. L-lysine hydroxamate,

b. S-(B-aminoethyl)-L-cysteine hydroxamate,

c. 3-(B-aminoethyl-sulfinyl) -L-alanine (S-dl),

d. 3-[S-methyl-S'(B-aminoethyl)-thiol-L-alamine,

e. S-B-( 2-pyridyl)ethyl-L-cysteine, and

f. S-B-(4-pyridyl)ethyl-L-cysteine, in a medium.

6 Claims, No lDrawings METHOD OF PRODUCINGL-LYSINE BY FERMENTATION FIELD or THE INVENTION v i BACKGROUND OF THE INVENTION It is well known that L-lysine is an indispensable amino acid for human and animal nutrition, and it is expected that L-lysine has a wide fuseas ah additive for the enrichment of food and foodstuffs.

Illustrative of prior processes for the preparation of L-lysine are the disclosures of UQS. Pat. No. 979,439 granted Apr l 1, 196.] and British Patent 1,258,380 granted Dec. .30, l9 7 l.. ln thelforrner casethe L- lysine producing bacteria is .a mutant which requires for its growth anamino acid .with a relationship to L-lysine biosynthesis such as a homoserine-requiringmutant of Micrococcusglutamicuss ln thelatter, case,..the L-lysine producing bacteria is a mutant which has aresistance to S-( Z-Amino ethyl) -L-cysteine, which is a sulfur analogue of L .-lysine. 4 l

It is an object of this.inventiontgproduce l lysine at a low cost from readily available raw materials.

DESCRIPTION ,OF THE INV ENTIQN The inventors havefoundthat lz lysine analogues which consist'of '-a. L'lysine hydr'oxamate, J I

b. S-(B-Amin'o' ethyU-L-c'ys'teine hydroxa'rnate d. B-[S-Methyl-S-(B-amino ethyl)-thio]-L-ala'riine,

e. S-B(2-Pyridyl)ethylLcysteine, and

f. S-B-(4-Pyridyl)ethyl-L-cysteine, have a physiological activity as L-lysine metabolic antagonist, and found that, when a mutant which has a resistance to one of thesemetabolic antagonists, is cultured in a suitable culturing medium, L-lysine is produced and accumulated in a large amount. The L'lysine produced is easily recovered from the medium.

The microorganism employed in this invention is a strain capable of producing L-lysine selected from mutants resistant to one of the above-mentionedL-lysine metabolic antagonists, and is easily obtained by mutant-inducing methods well known in the art from parental strains selected .from L-glutamic acid-producing microorganisms belonging to the genera Arthrobacter, Corynebacterium and Brevibacterium.

The microorganism employed in this invention hasa stable L-lysine producing activity, which is induced genet ically, and a decrease oflthis activity is not observed by back mutation. I

In addition; because the microorganismsemployed in this invention are strains which do not require for their growth a specific nutrient, the composition of the culturing medium employed in this invention is entirely conventional and economical. i i

-There have been many cases of overproduction of metabolites by such mutants, which are obtained as mutants resistant to the analogues of the objective metabolites. Overproduction ofLlysine by these mutants resistant to the analogues, might be due to the decrease of feedback inhibition and repression genetically.

. paraffins.

The culturing medium employed in this invention must contain an assimilable carbon source, an assimilable nitrogen source and the usual minor nutrients.

The carbon'sources suitable for this invention are carbohydrates such as glucose, molasses, starch hydrolysates, organic acids such as acetic acid, alcohols such as methyl alcohol and hydrocarbons such as n- The nitrogen sources suitable for this invention are ammonium salts of inorganic acids such as ammonium sulphate'and ammonium chloride, nitrate salts such as potassium nitrate, and sodium nitrate, ammonia in an aqueous solution or in the gaseous state, and urea.

The following is an experiment which shows the method of producing L-ly sine by this invention.

a. The Synthesis of L-Lysine Analogues We synthesized and obtained the next several new analogues of L-lysine. The analogues are listed in Table Compound a Compound b. Compound c 'CH Ni-l cH, NH erg-NH,

| v CH2 en, CH I I CH2 s =0 I g I. CH CH2 01 I I c i NH cH NH. I Y CH -NH l l CONHOH CONHOH coon Compound d Compound 0 Compound f N CH2-NH2 .CH. cu I /N s cH.. s 5 l t l l CH2 7 cu CH2 l l cH NH CH- NH (H-NH, l l l COOH coon coon methyl iodate.

Compound 2 was synthesized from triethylamine and 2-vinyl pyridine.

Compound f was synthesized from triethylamine and 4-vinyl pyridine. Y

The results are summarized in Table ll. As shown in Table 111, the six L-lysine analogues in- Table 11 Anal. Compound Culcd. Found c H N o c1 c H N o 5 c1 :1 38.41 8.53 22.40 17.07 13.59 38.35 8.23 22.84 17.08 13.50 b 27.84 6.50 19.48 14.83 14.87 16.48 27.80 6.10 19.92 14.76 15.01 16.41 c 27.71 6.05 12.93 22.15 14.80 16.36 27.49 608 12.90 22.41 14.77 1635 d 33.41 7.48 12.99 14.83 14.86 16.43 33.25 7.51 13.00 15.27 14.80 16.17 c 53.07 6.24 12.38 14.14 14.17 53.06 6.18 12.43 14.08 14.25 f 53.07 6.24 12.38 14.14 14.17 53.11 6.20 12.43 14.16 14.10

in order to test, whether these L-lysine analogues are L-lysine metabolic antagonists or not, two experiments consisting .of the growth inhibition by these L-lysine analogues and the recovery of the growth from inhibition were examined as follows. i

b. Growth Inhibition by L-lysine Analogues Effect of these L-lysine analogues on the growth of Arthrobacter SP 8-1 was estimatedl The growing cells were inoculated in medium A containing L-lysine analogues and L-threonine in various concentrations and the turbidity was estimated after shaking culture for 16 hrs. The growth responses were indicated by the relative growth which the maximum growth of unsupplemented control tubes is indicated as 100%.

Medium A glucose 30g, Urea 5g, K HPO lg, MgSO .7H O 0.5g, FeSO .7H O 10mg, MnSO .7H O 5mg, Biotin 57, Yeast extract 0.5g, L-threonine and L-lysine analogues various concentration in 1000ml distilled water at pH 7.0. The results are summarized in Table 111.

Table 111 mg/ml Concentration of Analogue mg/ml Concentration of L-threonine Relative growth Compound LII LII

KII

LII

LII

hibited the growth of Arthrobacter SP B-l.

c. Recovery of Growth from Inhibition Effect of L-lysine on the recovery of growth of Arthrobacter SP B-l from inhibition by these L-lysine analogues, was estimated. The growing cells were inoculated in medium containing L-lysine analogues, L- threonine and L-lysine in various concentration and the turbidity was estimated as the same method in (b) above-mentioned. The concentration of L-lysine necessary for recovery of growth from the inhibition was investigated. As shown in Table IV, L-lysine was effective in recovery of growth from inhibition by these L-lysine analogues.

From the data presented, it is apparent that these six L-lysine analogues are potent competitive inhibitors of L-lysine utilization and that these six L-lysine analogues are L-lysine metabolic antagonists. These data strongly suggested that L-lysine-producing mutants can be selected selecting for L-lysine analogue resistant mutants.

Table lV mg/ml mg/ml mg/ml Com- Concentration Concentration Concentration Relative pound of of of growth Analogue L-threonine L-lysine c1. Isolation of L-Lysine Analogue-Resistant Mutants O vernight cultured growing cells of Arthrobacter SP B-l or Corynebacterium SP 185 or Brevibacterium SP B-4 in medium B were washed and suspended in 0.1 M phosphate buffer.

Medium B Polypeptone g, Yeast extract 10g, NaCl 5g, in

l00Ornl tap water at pH 7.0 2 Then they were treated with N-methyl-N-nitro-N'- nitrosoguanidine in 0.1 M phosphate buffer at the concentration of 300'y/ml for 30 min. at 0 3C. Then washed and inoculated directly onto minimal medium supplemented with L-lysine analogue.

Medium C v r glucose g, (Ni-10 50 1.5g, urea l.5g, KH PO 1g, 1( l-1PQ -1g, MgSO .7H O 0.1 g, thiamine HCl l00'y, d-biotin 30y, L-lysine analogue lg, L- threonine lg, agar g, in 1000ml distilled water at pH 7.0.

Colonies appearing on the surface of agar plate during 2 7 days incubation were picked up as L-lysine analogue resistant mutants. For each L-lysine analogue, 500 resistant mutants were obtained.

e. Production of L-Lysine by L-Lysine Analogue Resistant Mutants Accumulation of L-lysine by these analogue resistant mutants was examined by incubation with aeration in 20ml of medium D at 30C for 72 hrs. in culture flasks. L-lysine produced was determined by the manometric method using lysine decarboxylase-of Bacterium cadaveris. Mutants that produced L-lysine monohydrochloride in goodyield are summarized in Table V.

Table V L-Lysinc Resistant Parent Resistant Y -FERM-P HCl to Mutant No. produced Compound strain No. ATCC NO. g/L

a Arthrohuctcr 1772-193 1295 23.8

0 SP B l 21868 501 1 17.3 1002 21.5 1798 12.1 a Brcvibactc 1443-54 1299 28.5

rium SP B-4 21864 1401 21.8 1405 10.2 1477 15.6 h Arthrobactcr 874-75 1302 41.5

SP B1 21861 349 i 23.4 1008 38.2 1067 37.6 301 15.7 b Corynebacte- 35123 (1301 39.8

rium SP 185 21862 391 18.9 1068 26.1 1 100 35.3 1306 32.9 b Brcvibactu- 1304-31 1303 42.7

rium SP B-4 21860 l 1306 11.2 c Arthrobactcr 55-8 1296 15.3

SP B-l 21867 67 i 13.8 80 14.9 33 12.2 72 14.7 c Brcvibacte- 1899-31 1298 18.1

Table V-Continued L-Lysine Resistant Parent Resistant FERM-P HCl to Mutant No. produced Compound strain No. ATCC NO. g/L rium SP B-4 1580 21865 14.8 1560 13.1 1922 16.9 1955 18.0 d Arthrobacter 1 162-166 1304 39.4

SP B-l 21859 417 38.4 1 24.3 1179 V 27.1 v 820 18.8 d Corynebacte- 1202-138 1306 42.4

rium S0 21857 1 137 40.7 1217 29.8 1227 31.0 1206 27.5 d Brevibacte- 1506-13 1297 32.8

rium SP B-4 21866 1501 29.7 1829 20.3 1834 18.6 1839 21.4 e Arthrobacter 269-51 1305 230 SP B-l 21858 6 16.5 127 12.1 362 10.7 493 13.5 f Corynebacte- 738-26 1 1300 19.0 rium SP 185 225 21863 12.4 341 17.2 755 13.3 791 13.1

As shown in Table V, it is apparent that these analogue resistant mutants have high L-lysine-producing ability.

f. The Recovery of L-Lysine from the Culture Broth The recovery of L-lysine from the culture broth may follow known methods. The culture broth is filtered to remove the cells. The filtrate is passed through a column of cation exchange resin. The resin column is eluted with aqueous ammonia, and L-lysine monohydrochloride crystalline is obtained under reduced pressure and a precipitation method.

The invention is illustrated by the following examples.

EXAMPLE 1 A fermentation medium was prepared to the following composition.

101 of the medium were introduced into 201 Jar Fermentor, and were sterilized at 1 10C for 30 min;

The medium was inoculated with the microorganisms shown in Table VI and incubated at pH 7.5 7.8, agitation rate 600 650 rpm, aeration rate ll/l/min., and temperature 30 32C.

7 8 After completion of the fermentation, L-lysine pro- EXAMPLE 3 duced was determined by Warburg method and recovr d by i ha pmcedure, A fermentation medium was prepared to the follow- The results are listed in Table VI. mg composltloni 5 Table VI Acetic acid 6 g/L Culturing L-Iysine'HCI L-lysinc'HCl gmgglg 2 Strain duration produced recovered Ammonium sun-me 5 H (hrs. g/L g IPO4 ,1 l0 M s0,-7H,o 0.5 Arthrobacter Biotin 50 Mg/l. SP 13-1 NO. 874-75 89 43.8 385 Thiamine hydrochloride 200 FERM-P No. 1302 Urea 1 g/L ATCC No. 21861 The medium prepared in the same way as mentioned Arthrobacter v SP 13.1 No. 1162-166 76 41,9 366 15 1n Example 1 was 1noculated with Brevlbactenum SP FERM-P 1304 B-4 No. 1304-31, FERM-P No. 1303, ATCC No. ATCC 21859 21860, and incubated for 76 hrs. Brevibacterium 7 After completion of the fermentation, L-lysine.HCl i 76 was in a concentration of 12.7 grams per liter of the R -P o. 7 ATCC NO. 21866 medlum- Arthrobacter I EXAMPLE 4 SP B-l N0. 269-51 72 35.5 288 FERMP Nov 1305 A fermentanon med1um was prepared to the follow- ATCC No. 21858 mg composition:

1 Corynebactcrium SP 188 No. 738-26 69 25.7

n-Paraffins 50 g/L FERMJ, 1300 Ammonium sulfate 5 ATCC NO. 21863 KHZPQ' 2 MgSO -7H O 1 Fes0,-7H. ,o 0.01 30 MnSO 7H O 0.01 caco 1 20 EXAMPLE 2 g N a pH i A fermentation medium was prepared to the follow- I ing composition, 1 20 ml of the med1um fwere lntroduced 1nto 500 ml flask, and were sterilized at 120C for 10 min. The me- 1 dium was inoculated with Arthrobacter SP B-] No. Cane molasses v 350 g/L 55-8, PERM-P NO. 1296, ATCC NO. 21867 and was Ammonium g 7 5 80 55 g/L incubated at 30C with agitation for 72 hrs. After completion of the fermentation, L-lysine'HCl was produced in a concentration of 4. 1 grams per liter of the medium.

The ATCC numbers appearing in the present specification and claims are the depository numbers under which the strains are deposited in the American Type The medium was treated in the same way as m'en- 40 tionedin'Example 1, except that it was inoculated with the microorganisms shown in Table VI].

Table V11 Culture Collection, Rockville, Md.A l1 restrictions on the availability to the public of these strains will be ir- Culmring mysincficl recovably removed upon the granting of the present pa- Struin :13:3 mfg/t tent and the deposits will be maintained to assure the permanent availability thereof to the public during the life of the present patent.

Corynebacterium SP 185 N01 351-23 83 33.8 It w1ll be obv1ous to those sk111ed in the art that vari- E ous changes may be made without departing from the .ATCC No.21862 sp1r1t of the 1nvent1on, and therefore the inventlon 1s Arthrobacter not limited to what is shown in the drawings and de- 2 Sign 7 89 scribed in the specification but only as indicated in the ATCC NO. 21868 pp Claims- What is claimed is: C b, -t :2 sz ggg 81 442 v 1. A method of produc1ng L-lys1ne by m1croorgan- Form-P No. 1306 1sms wh1ch compnses culturmg under aerobic cond1- ATCC No. 21857 tions, an L-lysinc-producing mutant selected from the Brcvihactcrium v group conslstlng of SP 13-4 No. 1899-31 94 I 22.1 Arthobacter B-l No. 1772193, FERM-P No. 1295, FERM-P NO. 12911 ATCC NO. 21868 ATC N .21865 C e Arthrobacter 13-1 N0. 55-8, FERM-P NO. 1296, Coryncbacteriurn 1 ATCC NO. 21867, 188 Ne 738-26 Brevibacterium B-4 NO. 150643, FERM-P N6. FERM-P Nov 1300 6 1 ATCC Noam 1297, ATCC No. 21866,

Brevibacterium B-4 No. 1899-31, FERM-P N0.

1298, ATCC NO. 28165, 7

Brevibacterium B-4 No.

1299, ATCC No. 21864,

Corynebacterium 18S No.

1300, ATCC No. 21863,

Corynebacterium 18S No.

1301, ATCC No. 21862,

Arthrobacter B-l No. 874-75, FERM-P No. 1302,

ATCC No. 21861,

Brevibacterium B-4, No. 1304-31, PERM-P No.

1303, ATCC No. 28160,

Arthrobacter B-l No. 1 162-166, FERM-P No. 1304,

ATCC No. 28159,

Arthrobacter B-l No. 269-51, PERM-P N0. 1305,

ATCC No. 28158, and

Corynebacterium 18S No. 1202-138, PERM-P No.

1306, ATCC No. 21857 and being resistant to one of the metabolic antagonists consisting of:

a. L-lysine hydroxamate b. S-(B-amino ethyl)-L-cysteine hydroxamate c. 3-(,8-amino ethyl-sulfinyl)-L-a1anine (S-dl) 1433-54, FERM-P No.

728-26, PERM-P No.

351-23, PERM-P No.

d. 3[S-methy1-S-( B-amino ethyl)-thio]-L-alanine e. S-B-(Z-pyridyl)ethyl-L-cysteine, and

f. S-B-(4-pyridyl)ethyl-L-cysteine in a medium containing at least one assimilable carbon source, a nitrogen source and inorganic and growthpromoting constituents, maintaining the pH value of the culturing medium within a range of from 5 to 8.5 until a substantial amount of L-lysine is accumulated in said medium and recovering L-lysine from the medium.

2. A method according to claim 1, wherein the assimilable carbon source is an organic acid or a salt thereof.

3. A method according to claim 1, wherein the assimilable carbon source is hydrocarbons.

4. A method according to claim 1, wherein the assimilable carbon source is alcohols.

5. A method according to claim 1, wherein the assimilable carbon source is a carbohydrate.

6. A method according to claim 1, wherein the assimilable nitrogen source is ammonia, an ammonium salt I UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION I PATENT NO. 1 3,905,867

DATED September 16, 1975 Page 1 of 2 INVENTOR( I Yasuo KURIMURA et a].

It is certified that error appears in the above-identified patent and that said Letters Patent hereby corrected as shown below:

[57] ABSTRACT, line I, delete L-alanine," and insert therefor -culturingline 9, "alamine" should read -alanine-- 1 Column 2, line 55, delete "H01" second occurrence Column 4, line 35, insert: --by-- after "selected" UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION PATENT NO. 3,905,867

DATED September 16, 1975 INVENTOR(S) KURIMURA et a1 It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Page 2 of 2 Column 8, line 67 should read --l298, ATCC No. 21865,-- Column 9, line 10 should read -l303, ATCC No. 2l860,-

' line 12 should read --ATCC No. 21859,--

" line 14 should read --ATCC No. 21858, and- Q Signed and Striated this seventeenth Day Of February 1976 [SEAL] Attest:

RUTH C. MASON C. MARSHALL DANN Arresting Officer Commissioner vfPatenrs and Trademarks 

1. A METHOD OF PRODUCING L-LYSINE BY MICROORGANISMS WHICH COMPRISES CULTURING UNDER AEROBIC CONDITIONS, AN LLYSINE-PRODUCING MUTANT SELECTED FROM THE GROUP CONSISITNG OF ARTHOBACTER B-1 NO. 1772-193, FERM-P NO. 1295, ATCC NO. 21868, ARTHOBACTER B-1 NO. 55-8, FERM-P NO. 1296, ATCC NO. 21867, BREVIBACTERIUM B-4 NO. 1506-13, FERM-P NO. 1297, ATCC NO. 21866, BREVIBACTERIUM B-4 NO. 1899-31, FERM-P NO. 1298, ATCC NO. 28165, BREVIBACTERIUM B-4 NO. 1433-54, FERM-P NO. 1299, ATCC NO. 21864, CORYNEBACTERIUM 18S NO. 728-26, FERM-P NO. 1300, ATCC NO. 21863, CORYNEBACTERIUM 18S NO. 351-23, FERM-P NO. 1301, ATCC NO. 21862, ARTHROBACTER B-1 NO. 874-75, FERM-P NO. 1302, ATCC NO. 21861, BREVIBACTERIUM B-4, NO. 1304-31, FERM-P NO. 1303, ATCC NO. 28160, ARTHROBACTER B-1 NO. 1162-166, FERM-P NO. 1304, ATCC NO. 28159, ARTHROBACTER B-1 NO. 269-51, FERM-P NO. 1305, ATCC NO. 28158, AND CORYNEBACTERIUM 18S NO. 1202-138, FERM-P NO. 1306, ATCC NO. 21857 AND BEING RESISTANT TO ONE OF THE METABOLIC ANTAGONISTS CONSISTING OF: A. L-LYSINE HYDROXAMATE B. S-(B-AMINO ETHYL)-L-CYSTEINE HYDROXAMATE C. 3-(B-AMINO ETHYL-SULFINYL)--L-ALANINE (S-DL) D. 3(S-METHYL-S-(B-AMINO ETHYL)-THIO)-L-ALANINE E. S-B-(2-PYRIDYL)ETHYL-L-CYSTEINE, AND F. S-B-(4-PYRIDYL)ETHYL-L-CYSTEINE IN A MEDIUM CONTAINING AT LAST ONE ASSIMILABLE CARBON SOURCE, A NITROGEN SOURCE AND INORGANIC AND GROWTH-PROMOTING CONSTITUENTS, MINTAINING THE PH VALUE OF THE CULTURING MEDIUM WITHIN A RANGE OF FROM 5 TO 8.5 UNTIL A SUBSTANTIAL AMOUNT OF L-LYSINE IS ACCUMULATED IN SAID MEDIUM AND RECOVERING LLYSINE FROM THE MEDIUM.
 2. A method according to claim 1, wherein the assimilable carbon source is an organic acid or a salt thereof.
 3. A method according to claim 1, wherein the assimilable carbon source is hydrocarbons.
 4. A method according to claim 1, wherein the assimilable carbon source is alcohols.
 5. A method according to claim 1, wherein the assimilable carbon source is a carbohydrate.
 6. A method according to claim 1, wherein the assimilable nitrogen source is ammonia, an ammonium salt or urea. 